A Method for Programmatically Managing Antibody Disulfide Bonds Site-Specific Modification

This application claims the priority to PCT Application No. PCT/CN2022/113992, filed on Aug. 22, 2022, PCT Application No. PCT/CN2022/119999, filed on Sep. 20, 2022, PCT Application No. PCT/CN2022/119955, filed on Sep. 20, 2022, PCT Application No. PCT/CN2022/131519, filed on Nov. 11, 2022, PCT Application No. PCT/CN2022/131521, filed on Nov. 11, 2022, and PCT Application No. PCT/CN2023/073070, filed on Jan. 19, 2023.

The contents of the prior PCT applications are considered as a part of the present disclosure and are incorporated herein in its entirety.

The disclosure relates to a method for programmatically managing antibody disulfide bonds site-specific modification, the modified antibody prepared by the method and the use thereof.

The statements in this section merely provide background information related to the present disclosure and do not necessarily constitute prior art.

Site-specific modification approaches have been extensively employed in the development of protein-based technologies. The modification of proteins has emerged as a valuable approach to interrogate and to intervene in biological systems. A protein modification approach, without altering the structure or activity of the protein, is crucial to the development of protein-drug conjugates, including antibody-drug conjugates (ADCs).

Generally, antibody conjugation to cytotoxic agents commonly involves conjugation to exposed residues including lysines or reduction of disulfide bonds to expose free interchain cysteines on a therapeutic IgG (Immunoglobulin G) antibody. There are other, more recent approaches that introduce conjugation sites to the mAb such as site-specific glycan conjugation, cysteine engineering, incorporation of unnatural amino acids and coupling short peptide tags to drug-linkers. There are typically 80 lysine residues on an antibody; however, less than ten residues are chemically accessible for conjugation. Cysteine conjugation eventuates in the reduction of four interchain disulfide bonds. These bonds are reduced under specific conditions and subsequently result in two, four, six or eight exposed sulfhydryl groups. Both Cys and Lys conjugation methods result in heterogeneous mixtures. (“Advances and Limitations of Antibody Drug Conjugates for Cancer”. Biomedicines. 2021 August; 9(8): 872.).

The drug-antibody ratio (DAR), or number of drug molecules conjugated to a single ADC, is very important for the determination of efficacy of ADCs. DAR widely varies and depends on other ADC variables. The DAR values are also dependent on the site of conjugation and the use of light or heavy conjugated chains. The DAR value influences the effectiveness of the medicine due to the depression in potency caused by low drug loading, while elevated drug loading can impact toxicity and pharmacokinetics (“Introduction to Antibody-Drug Conjugates”. Antibodies (Basel). 2021 December; 10(4): 42.).

A number of methods have been developed to improve the homogeneity of ADCs. For example, Synaffix's technology GlycoConnect™ (US2015/0320882, synaffix.com/platform/technology/) has been developed to covert an antibody into a stably conjugated ADC with DAR2, DAR4 or even DAR1 and DAR6, by modifying the native antibody glycan through a three-step process: enzyme digestion, enzyme mediated ligation and metal-free click chemistry

US20210040145 discloses a 14-amino acid peptide Tub-tag fused to the C-terminus of any protein of interest (POI) and catalyzes the addition of a variety of different tyrosine derivatives. Taking advantage of this enzyme, Tub-tag technology repurposed tubulin-tyrosine ligase for the attachment of functional moieties at the C-terminus of antibody to homogeneously generate antibody conjugates with DAR 2.

However, those technologies suffer from several drawbacks, such as immunogenicity risk, complicated purification, and/or high cost.

Therefore, there is still a need for site-specific modification of an antibody.

For the above-mentioned purpose, provided herein is a method for programmatically managing antibody disulfide bonds site-specific modification, comprising step,

In some embodiments, in step (R1), one of the interchain disulfide bond of the antibody is reduced, optionally, one of the interchain disulfide bond in the hinge region of the antibody is reduced.

In some embodiments, in step (R1), three of the interchain disulfide bonds of the antibody are reduced, optionally, two of the interchain disulfide bonds in the Fab region and one of the interchain disulfide bond in the hinge region of the antibody are reduced.

In some embodiments, the method also includes step (O1),

In some embodiments, the method also includes step (R2),

In some embodiments, the method further comprises the following step,

In some embodiments, the method further comprises the following step,

In some embodiments, the method also includes the following steps,

The method provided herein is compatible with current thiol-reactive linker-drug technologies with minimum conformation change and intact Fc function. Meanwhile it has simple manipulation and reduced cost and is simple to operate without enzymes engineering, and it is fully compatible with current thiol-reactive linker-drug technologies.

In one aspect, provided herein is a modified antibody prepared by the method above. The modified antibody is conjugated with one, two or three kinds of conjugating groups.

In one aspect, provided herein is a pharmaceutical composition comprising the modified antibody prepared by the method above, and at least one pharmaceutically acceptable ingredient.

In one aspect, provided herein is the use of the modified antibody prepared by the method above or the pharmaceutical composition provided herein in the manufacture of a therapeutic agent for preventing, diagnosing, or treating a disease.

In one aspect, provided herein is a method of preventing or treating a disease in a subject in need thereof, comprising administrating to the subject a therapeutically effective amount of a modified antibody prepared by the method above.

The present disclosure is explained in greater detail below. This description is not intended to be a detailed catalog of all the different ways in which the invention may be implemented, or all the features that may be added to the instant invention. For example, features illustrated with respect to one embodiment may be incorporated into other embodiments, and features illustrated with respect to a particular embodiment may be deleted from that embodiment. In addition, numerous variations and additions to the various embodiments suggested herein will be apparent to those skilled in the art in light of the instant disclosure which do not depart from the instant invention. Hence, the following description is intended to illustrate some particular embodiments of the invention, and not to exhaustively specify all permutations, combinations and variations thereof.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the disclosure pertains. Although any methods and materials similar or equivalent to those described herein may be used in the practice for testing of the present disclosure, the preferred materials and methods are described herein. In describing and claiming the present disclosure, the following terminology will be used.

Unless otherwise defined herein, scientific and technical terms used in connection with the present disclosure shall have the meanings that are commonly understood by those of ordinary skill in the art. Further, unless otherwise required by context, singular terms shall include pluralities and plural terms shall include the singular. More specifically, as used in this specification and the appended claims, the singular forms “a,” “an” and “the” include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to “an antibody” includes a plurality of antibodies; reference to “a transition metal ion” includes mixtures of transition metal ions, and the like. In this application, the use of “or” means “and/or” unless stated otherwise.

Throughout this disclosure, unless the context requires otherwise, the words “comprise”, “comprises”, “comprising”, “contain”, “contains” and “containing” will be understood to imply the inclusion of a stated step or element or group of steps or elements but not the exclusion of any other step or element or group of steps or elements. By “consisting of” is meant including, and limited to, whatever follows the phrase “consisting of”. Thus, the phrase “consisting of” indicates that the listed elements are required or mandatory, and that no other elements may be present.

Provided herein is a method for programmatically managing (controlling) antibody disulfide bonds modification, which could modify a protein, such as an antibody, to covalent link a group of interest. The products may be antibody-drug conjugates (ADCs), groups of interest (e.g., cytotoxic drug) linking to a monoclonal antibody via linkers or thiobridge reagents. Particularly, provided herein is a method for programmatically managing antibody disulfide bonds site-specific modification, the site-specific modified antibody with improved homogeneity are benefit for enhancing safety and curative effect.

The present disclosure provides examples of methods for programmatically managing antibody disulfide bonds site-specific modification, the method includes the following step,

The reductant, optionally, with the transition metal ions, has reducibility and reduces the disulfide bond of an antibody to modify protein or antibody. Optionally, the reductant selectively reduces the interchain disulfide bonds, thus the antibody is selectively modified.

In some embodiments, provided herein is the method I or method II for programmatically managing antibody disulfide bonds site-specific modification. In some embodiments, in step (R1) of method I, one of the interchain disulfide bond of the antibody is reduced, optionally one of the interchain disulfide bond in the hinge region of the antibody is reduced. In some embodiments, in step (R1) of method II, three of the interchain disulfide bonds of the antibody are reduced, optionally two of the interchain disulfide bonds in the Fab region and one of the interchain disulfide bond in the hinge region of the antibody are reduced.

In some embodiments, the method I and/or method II includes the following steps,

In some embodiments, in step (C1) of method I, one or two of the first conjugating groups are covalently linked to the reduced thiol groups resulted from step (R1).

In some embodiments, in step (C1) of method II, three or six of the first conjugating groups are covalently linked to the reduced thiol groups resulted from step (R1).

As used herein, the term “disulfide bond” refers to a covalent bond with the structure R—S-S-R′. The amino acid cysteine comprises a thiol group that can form a disulfide bond with a second thiol group, for example from another cysteine residue. The disulfide bond can be formed between the thiol groups of two cysteine residues residing respectively on the two polypeptide chains, thereby forming an interchain bridge or interchain bond.

As used herein, the term “hinge region” refers to an antibody includes the portion of a heavy chains molecule that joins the CH1 domain to the CH2 domain. This hinge region comprises approximately 25 amino acid residues and is flexible, thus allowing the two N-terminus antigen binding regions to move independently.

As used herein, the term “Fab fragments” refers to the region of the antibody structure that can bind to antigen. It consists of a complete light chain (variable and constant regions) and part of the heavy chain structure (variable and a constant region fragment), the light and heavy chains are connected by a disulfide bond. Fab fragments can be obtained by protease digestion of full-length antibodies. Under the action of papain, human immunoglobulin G can be degraded into two Fab fragments and one Fc fragment; under the action of pepsin, IgG can be degraded into an F(ab′)2 fragment and a pFc′ fragment. The F(ab′)2 fragment can be further reduced to form two Fab′ fragments. In some embodiments, the interchain disulfide bonds connect two of the upper heavy chains in the hinge region or the interchain disulfide bonds connect the heavy chain to the light chain in Fab region.

As used herein, the term “antibody” refers to any immunoglobulin, monoclonal antibody, polyclonal antibody, multispecific antibody, or bispecific (bivalent)antibody that binds to a specific antigen. A native intact antibody comprises two heavy chains and two light chains. Each heavy chain consists of a variable region (“HCVR”) and a first, second, and third constant region (CH1, CH2 and CH3), while each light chain consists of a variable region (“LCVR”) and a constant region (CL). Mammalian heavy chains are classified as α, δ, ε, γ and μ, and mammalian light chains are classified as λ or κ. The antibody has a “Y” shape, with the stem of the Y consisting of the second and third constant regions of two heavy chains bound together via disulfide bonding. Each arm of the Y includes the variable region and first constant region of a single heavy chain bound to the variable and constant regions of a single light chain. The variable regions of the light and heavy chains are responsible for antigen binding. The variable regions in both chains generally contain three highly variable loops called the complementarity determining regions (CDRs) (light (L) chain CDRs including LCDR1, LCDR2, and LCDR3, heavy (H) chain CDRs including HCDR1, HCDR2, HCDR3). CDR boundaries for antibodies may be defined or identified by the conventions of Kabat, Chothia, or Al-Lazikani (Al-Lazikani, B., Chothia, C., Lesk, A. M., J. Mol. Biol., 273(4), 927 (1997); Chothia, C. et al., J Mol Biol. December 5; 186(3): 651-63 (1985); Chothia, C. and Lesk, A. M., J. Mol. Biol., 196, 901 (1987); Chothia, C. et al., Nature. December 21-28; 342(6252): 877-83 (1989); Kabat E. A. et al., National Institutes of Health, Bethesda, Md. (1991)). The three CDRs are interposed between flanking stretches known as framework regions (FRs), which are more highly conserved than the CDRs and form a scaffold to support the hypervariable loops. Each HCVR and LCVR comprises four FRs, and the CDRs and FRs are arranged from amino terminus to carboxy terminus in the order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The constant regions of the heavy and light chains are not involved in antigen binding, but exhibit various effector functions. Antibodies are assigned to classes based on the amino acid sequence of the constant region of their heavy chain. The five major classes or isotypes of antibodies are IgA, IgD, IgE, IgG, and IgM, which are characterized by the presence of α, δ, ε, γ, and μ heavy chains, respectively. Several of the major antibody classes are divided into subclasses such as IgG1 (γ1 heavy chain), IgG2 (γ2 heavy chain), IgG3 (γ3 heavy chain), IgG4 (γheavy chain), IgA1 (α1 heavy chain), or IgA2 (α2 heavy chain).

In some embodiments, the method comprises step (O1),

In some embodiments, provided herein is the method III for programmatically managing antibody disulfide bonds site-specific modification. The method III includes the following steps,

In some embodiments, the oxidant in step (O1) re-oxidizes the reduced thiol groups in Fab region of the antibody resulted from step (R1), optionally, four of the reduced thiol groups are re-oxidized to form two disulfide bonds in step (O1).

In some embodiments, in step (C1) of method III, one or two of the first conjugating groups are covalently linked to the remaining thiol groups resulted from step (O1).

In some embodiments, the method comprises step (R2),

In some embodiments, provided herein is the method IV for programmatically managing antibody disulfide bonds site-specific modification, the method IV includes the following steps,

In some embodiments, in step (R2) of method IV, one of the interchain disulfide bond in the hinge region of the antibody is reduced.

In some embodiments, in step (C1) of the method IV, two or four of the first conjugating groups are covalently linked to the remaining thiol groups resulted from step (R2).

In some embodiments, the method further comprises the following step, (C2) incubating at least equimolecular proportion of the first conjugating group to react with the reduced thiol groups resulted from step (R1), calculated as the molar amount of antibody, then, optionally, introducing the metal chelators and the oxidant, or optionally, introducing the metal chelators and the first conjugating group.

In some embodiments, provided herein is the method V for programmatically managing antibody disulfide bonds site-specific modification, the method V includes the following steps,

In some embodiments, provided herein is the method V for programmatically managing antibody disulfide bonds site-specific modification, the method V includes the following steps,